Author ORCID Identifier

https://orcid.org/0000-0002-9612-4803

Semester

Spring

Date of Graduation

2023

Document Type

Dissertation

Degree Type

PhD

College

Davis College of Agriculture, Natural Resources and Design

Department

Animal and Nutritional Sciences

Committee Chair

Jianbo Yao

Committee Member

Melanie Clemmer

Committee Member

Robert Dailey

Committee Member

Daniel Mathew

Committee Member

I-Chung Chen

Abstract

The oocyte expresses certain genes during folliculogenesis to regulate the acquisition of oocyte competence. Oocyte competence, which refers to the presence of imperative molecular factors in the oocyte that are critical for high oocyte quality, is directly related to the ability of the oocyte to result in a successful pregnancy following fertilization. Over the past few decades, the development and optimization of assisted reproductive technologies, particularly in vitrofertilization, have enabled the beef and dairy industries to advance cattle genetics and productivity. However, only approximately 40% of bovine embryos will develop to the blastocyst stage in vitro. In addition, bovine embryos produced in vitro are developmentally inferior compared to in vivo derived embryos due to the lack of optimization of the oocyte and embryo culture conditions in vitro. Characterization of factors regulating these processes is crucial to improve the efficiency of bovine in vitro embryo production.

RNA Sequencing data obtained by our laboratory demonstrated that the secreted protein, agouti-signaling protein (ASIP), is highly abundant in the bovine oocyte. Agouti-signaling protein (ASIP) has a characterized role in the distribution of melanin pigment in some mammalian species, including mice. In adipose tissue, ASIP expression is associated with insulin resistance and obesity. Recently, it was demonstrated that ASIP is crucial in regulating mammary epithelial cell lipid metabolism in cattle. However, the role of ASIP in the bovine oocyte and early embryo has not been previously elucidated. This research aimed to characterize the ASIP spatiotemporal expression profile in the ovary and throughout early embryonic development. Further, objectives included revealing the effects of supplementation of ASIP during in vitro oocyte maturation and embryo culture on subsequent embryonic development.

In addition to oocyte expression, ASIP was detected in granulosa, cumulus, and theca cells isolated from antral follicles. Both ASIP mRNA and protein were found to decline with oocyte maturation, suggesting a prospective role for ASIP in achieving oocyte competence. Microinjection of presumptive zygotes using small interfering RNAs targeting ASIP led to a 13% reduction in the rate of development to the blastocyst stage. Additionally, we examined potential ASIP signaling mechanisms through which ASIP may function to establish oocyte developmental competence. Expression of melanocortin receptors 3 and 4 and the coreceptor attractin was detected in the oocyte and follicular cells. Interestingly, the addition of cortisol, which was previously determined to be beneficial for oocyte competence in cattle, during in vitro maturation significantly increased oocyte ASIP levels.

Cumulus-oocyte complexes or presumptive zygotes were placed in culture medium containing either 0, 1, 10, or 100 ng/mL of recombinant ASIP, and effects on subsequent development, gene expression, lipid content, and blastocyst cell allocation were examined. Supplementation of ASIP during oocyte maturation improved the blastocyst development rate and produced blastocysts with an increased inner cell mass to trophectoderm cell ratio. Nile red staining revealed that adding ASIP during oocyte maturation increased oocyte but not embryo lipid levels. The expression of genes involved in lipid metabolism, including FASN, PPAR𝛾, SCD, CSL1, ELOVL5, and ELOVL6, were not found to be significantly altered in blastocysts due to treatment. Meanwhile, supplementation of ASIP during embryo culture did not affect blastocyst rates.

These results support a functional role for ASIP in promoting oocyte maturation and subsequent embryonic development, potentially through signaling mechanisms involving cortisol. Additionally, these data further support the role of ASIP in acquiring oocyte competence and suggest that supplementing ASIP during oocyte maturation may lead to the production of blastocysts of increased quality. Future prospective applications of this work include optimizing bovine oocyte or embryo culture conditions to emulate better the in vivo maternal environment through normalizing lipid metabolism and, subsequently, minimizing stress. Further, future research should explore the utilization of ASIP in developing improved cryopreservation techniques for bovine embryos.

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